Characterization and possible mechanisms of alpha 2-adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production in HT29 cells.

S B Jones,D B Bylund

doi:10.1016/s0021-9258(18)68211-0

Abstract

Preincubation with an alpha 2-adrenergic agonist sensitized subsequent forskolin- and vasoactive intestinal peptide-stimulated cyclic AMP production in HT29 cells, a human colonic adenocarcinoma cell line. Preincubation with somatostatin, another agonist negatively coupled to adenylate cyclase, sensitized forskolin-stimulated cyclic AMP production to a lesser extent. alpha 2-Adrenergic agonist preincubation also resulted in desensitization as indicated by a shift to the right in the dose-response curve of a subsequent challenge by an alpha 2-adrenergic agonist. In an effort to elucidate the mechanism for sensitization, we examined protein kinase C and the Na+/H+ antiporter. Whereas these components had marked effects on forskolin stimulation, there was no effect on sensitization. Changes in the concentration of extra-cellular Ca2+ or Mg2+ had no effect on either forskolin stimulation or sensitization. Pertussis toxin pretreatment caused a time-dependent decrease in sensitization, an attenuation of inhibition of cyclic AMP production, and a decrease in subsequent [32P]ADP-ribosylation by pertussis toxin. The time course for these three events was similar, implicating the inhibitory guanine nucleotide regulatory protein in the mechanism for alpha 2-adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production. In addition, pertussis toxin dramatically decreased forskolin-stimulated cyclic AMP production, although with a different time course. These results suggest that the mechanism of sensitization is via an as yet undefined sequence of biochemical events that includes the inhibitory guanine nucleotide regulatory protein, but does not include inhibition of adenylate cyclase nor activation of the Na+/H+ antiporter.

Highlights

Preincubation with an a2-adrenergic agonist sensi- HT29 cells, sensitization is rapid in onset and reversal, and tized subsequent forskolin- and vasoactive intestinal is mediated via the a2-adrenergicreceptor
We have recently reported that preincubation with an azadrenergic agonist sensitizes subsequent forskolin-stimulated adenylate cyclase, implicating protein kinase C in this event [11, 12].Theattenuation of inhibitionin the platelet by cyclic AMP production in HT29 cells, a human colonic ade- phorbol ester treatment is correlated with the phosphorylanocarcinoma cell line [1].An apparently similar phenomenon tion of Gi, further implicating protein kinase C intervention has beenobserved in several receptor systems which are [13]
We have examined the specificity of sensitization in HT29 cells and report that somatostatin preincubation sensitizes forskolin-stimulated cyclic AMP production, the degree of sensitization is much lower. a,Adrenergic agonist preincubation results in sensitization of vasoactive intestinal peptide (VIP)-stimulated cyclic AMP production which is lower in magnitude and perhapsalso different in mechanism than forskolin sensitization.In an effort to elucidate the mechanism for sensitization, we have examined protein kinase C, the Na+/H+antiporter, changes in extracellular cation concentration, andthe guanine nucleotide regulatory protein, Gi

Summary

EXPERIMENTAL PROCEDURES

Cell Culture"HT29 cells, a human colonic adenocarcinoma cell line, were obtained from J. 50-100 pg of protein were incubated with 1.2 mM [w3*P]ATPin 75 pl of a medium containing 50 mM Tris-HC1 (pH 7.6), 6.7 mM MgC12, 25 mM creatine phosphate, 5 units of creatine phosphokinase, 1 mM cyclic AMP, 10 p M GTP, 30 mM NaCl, 0.7 mM isobutylmethylxanthine, and 2 mg/ml bovine serum albumin. The pellet was suspended for 10 min in ice-cold suspension buffer consisting of 5 mM Tris (pH 7.5) containing 1mM each dithiothreitol and EDTA. 70 fig of protein were incubated at 30 "C for 50 min with 6.5 p~ [32P]NADin the presence of 2.5 pg ofpertussis toxin in areaction mixture consisting of 100 mM potassium phosphate buffer (pH 7.5), 10 mM thymidine, 1mM ATP, 0.5 mM EDTA, and 20 mM dithiothreit in a final volume of 100 al. The followingdrugs were graciously donated by the respective companies: UK14,304 (Pfizer, Groton, CT), 1-propranolol hydrochloride (Ayerst Laboratories, NY)

RESULTS

Sodium BSS Choline BSS

We have shownpreviously that sensitization is rapid in onset

TIME PRETREATMENT